The analysis of microorganisms in a liquid, such as water, milk, or soft drinks for human consumption and water for microelectronic and pharmaceutical product processing is of the utmost importance to health and product integrity, respectively. Microbial count analysis typically consists of a labor intensive manual membrane filtration followed by a 48 to 72 hour incubation period. Several different growth media and incubation temperatures are often necessary to get a full microbial profile of a particular liquid.
The results of the microbial analysis are interpreted by a trained analyst who visually counts the number of colonies on the filter paper after incubation. Each colony represents a single colony forming unit (CFU) in the initial sample. From this information, the number of CFUs per unit volume is determined.
The long lag times of 48 to 72 hours required for a microbial analysis frequently lead to product waste in the microprocessor and pharmaceutical industries since a failure requires that products already produced, have to be discarded. A chronic microbial contamination situation can lead to 48-72 hours worth of discarded products before testing alerts the user to the unacceptable situation.
Further, the capability for microbial monitoring of processed liquid for human consumption is necessary for health and welfare. Membrane filtration, as previously stated, is labor intensive, is not selective to all potentially present microbes as a single test, and required 48-72 hours of incubation time prior to acquiring results. With a 48-72 hour lag time before results, for example, a space station user is faced with the dilemma of deciding whether the test results of a 2-3 day old sample are still representative of the actual water source.
Bioluminescence has been utilized by both the food industry and medical field to detect high concentrations of microbes. The detection process consists of measuring adenosine triphosphate (ATP); a nucleotide found in all living cells. ATP, the primary energy donor in viable cells, rapidly degrades as the cell dies. With the use of the enzyme luciferase (see U.S. Pat. No. 4,833,075), ATP can be measured.
ATP is released from living bacterial cells with the use of a bacterial release agent which lyses microbial cell walls. In the presence of oxygen (O.sub.2), magnesium (Mg.sup.++) and luciferase, ATP drives the conversion of luciferin to oxyluciferin. This reaction results in the conversion of ATP to adenosine monophosphate (AMP) and the release of a photon of light. ##STR1## The light can be measured by a luminometer if the concentration of microbes is about 80-100 CFU absolute or greater, the current sensitivity for the state of the art. Note, bioluminescence is typically utilized for grossly concentrated samples, not for trace level analysis.
This process is typically accomplished by sequentially adding a bacterial release agent, luciferin/luciferase, and Mg.sup.++ to a liquid sample while a fixed luminometer records any offsets due to emitted light from the sample.
At present, bioluminescence is used to monitor growth enriched samples of filtered liquids. For example, U.S. Pat. No. 5,141,869 is directed to a microbial monitor having a bladder for growth enrichment of microbial concentrations in a liquid sample with a growth buffer and incubation for enriching the amount of microbes in the liquid sample.
Each of the above discussed analysis of microorganisms in liquids requires a complicated sampling system requiring long periods of time to incubate a sample, e.g. in an incubator and using a growth buffer, prior to count analysis.